Insect species in the genus Diabrotica (corn rootworms and cucumber beetles) are considered some of the most important pests to crop plants. For example, species of corn rootworm, including Diabrotica virgifera virgifera, the western corn rootworm (WCR); D. barberi, the northern corn rootworm (NCR), D. undecimpunctata howardi, the southern corn rootworm (SCR), and D. virgifera zeae, the Mexican corn rootworm (MCR), are the most destructive corn pests in North America causing an estimated loss of over $1 billion annually. The western corn rootworm has also invaded Europe and causes an estimated 0.5 billion euros in damage each year. Diabrotica speciosa (common names include, among others, leaf beetle, little Brazilian beetle, cucurbit beetle and chrysanthemum beetle) is an important pest of corn, soybean and peanuts, in South America.
Most of the damage in corn is caused by larval rootworm feeding. Newly hatched rootworm larvae locate corn roots in the soil and initially begin feeding on the fine root hairs and burrow into root tips of the corn plant. As larvae grow larger, they feed on and tunnel into primary roots. When rootworms are abundant, larval feeding and deterioration of injured roots by root rot pathogens can result in roots being pruned to the base of the stalk. Severe root injury interferes with the roots' ability to transport water and nutrients into the plant, reduces plant growth, and results in reduced grain production. Severe root injury also may result in lodging of corn plants, making mechanical harvest more difficult or impossible. Corn rootworm adults feed primarily on corn silk, pollen, and kernels on exposed ear tips. If corn rootworm adults begin emerging before corn reproductive tissues are present, adults may feed on leaf tissue, scraping away the green surface tissue and leaving a window-pane appearance. Silk feeding by adults can result in pruning of silks at the ear tip, commonly called silk clipping. In field corn, beetle populations may reach a level high enough to cause severe silk clipping during pollen shed, which may interfere with pollination and reduce yield. Thus, unlike lepidopteran pests of corn in which only the larval stages cause damage, both the larval and adult stages of corn rootworm are capable of causing economic damage to corn.
Diabrotica insect pests are mainly controlled by intensive applications of chemical pesticides, which may be active against both larval and adult stages, through inhibition of insect growth, prevention of insect feeding or reproduction, or cause death. Good insect control can thus be reached, but these chemicals can sometimes also affect other, beneficial insects. Additional problems occur in areas of high insecticide use where populations of corn rootworm beetles have become resistant to certain insecticides. This has been partially alleviated by various resistance management practices, but there is an increasing need for alternative pest control agents.
Several native Cry proteins from Bacillus thuringiensis, or engineered Cry proteins, have been expressed in transgenic crop plants and exploited commercially to control certain lepidopteran and coleopteran insect pests. For example, starting in 2003, transgenic corn hybrids that control corn rootworm by expressing a Cry3Bb1, Cry34Ab1/Cry35Ab1 or modified Cry3A (mCry3A) protein have been available commercially in the US.
The seed industry, university researchers and the US Environmental Protection Agency have worked together to develop management plans to help mitigate the onset of insect resistance to transgenic plants expressing insecticidal proteins. They are based primarily on a high dose and refuge strategy. A high dose strategy is to use corn hybrids that express high enough levels of an insecticidal protein such as a Cry protein to kill even partially resistant insects. The underlying hypothesis is that killing partially resistant insects and preventing their mating greatly delays the development of resistance. The success of a high dose strategy depends in part on the specific activity of the insecticidal protein to the particular insect species and how much of that insecticidal protein can be expressed in the transgenic corn plant. The higher the specific activity of an insecticidal protein to a pest, the less amount of the insecticidal protein is required to be expressed in a transgenic plant to achieve a high dose strategy. For example, corn hybrids expressing the lepidopteran-active Cry protein, Cry1Ab, are considered high-dose against the primary target pest European corn borer (Ostrinia nubilalis). Because Cry1Ab is very toxic to European corn borer larvae with an LC50<10 ng/cm2 (i.e. high specific activity), levels of expression of Cry1Ab that are achievable in transgenic plants easily places such corn hybrids in a high dose category. However, unlike the lepidopteran-active products, current rootworm products are not considered high-dose. The proteins they express are not active against adults and have limited activity against late instar larvae. Therefore, the current transgenic rootworm products allow some rootworm larvae to survive and emerge as adults.
Thus, economic levels of silk clipping by corn rootworm adults may still occur even in portions of fields planted to a transgenic corn rootworm hybrid. For example, densities of western corn rootworm adults may exceed economic levels in portions of fields planted to transgenic corn rootworm hybrids due to immigration of beetles as well as direct emergence of adults from transgenic root systems. There have been many reports that confirm western corn rootworm adult emergence from certain corn transgenic rootworm hybrids (Crowder et al. 2005. J. Econ. Entomol. 8:961-975). A recent publication suggests that western corn rootworm adults will exhibit similar feeding behaviors when encountering some transgenic corn plants or non-transgenic corn plants in the field and that it is unlikely that certain insecticidal proteins in transgenic plants will have significant effects on adults that might impact resistance management.
Therefore, indentifying alternative insect control agents with new modes of action would be beneficial. Particularly useful would be new insect control agents that are toxic to multiple life stages of the target insect pest. Such insect control agents may include those that target genetic elements in the target insect pest.
Apoptosis is a physiological cell death process that is critical for the development and maintenance of healthy biological systems in many living organisms. The Inhibitor of Apoptosis (IAP) family of proteins was first discovered in baculoviruses that attack insects and have been shown to be involved in suppressing the insect host cell death (apoptosis) response to baculovirus infection (Crook et al. 1993. J. Virol. 67:2168-2174; Birnbaum et al. 1994. J. Virol. 68:2521-2528). Subsequently, IAP proteins were discovered in other organisms including the yeast Saccharomyces cerevisiae, the nematode Caenorhabditis elegans, the fly Drosophila melanogaster, and several mammalian species. Native IAP acts as an endogenous inhibitor of caspases, the main executioners of apoptosis and IAP family proteins are characterized by an approximately 70 amino acid domain termed the baculoviral IAP repeat (BIR). Up to three tandem copies of the BIR domain can occur within the known IAP family proteins of viruses and animal species. Although membership in the IAP family of proteins requires both the presence of a BIR domain and the ability to suppress apoptosis, many of these BIR-containing proteins are untested with respect to apoptosis suppression. Moreover, as it is debatable as to whether yeast possess an apoptosis program (Zha et al. 1996. Mol Cell Biol. 16:6494-6508), because the presence of BIR-containing proteins in S. pombe and S. cerevisiae raises the possibility that BIR domains are not devoted exclusively to apoptosis suppression. Rather, they may function as protein interaction domains that may have evolved to suit a variety of purposes.
Therefore, it is not clear that all IAPs are part of life-critical pathways for any given organism, particularly in certain insect species including coleopteran pest species like Diabrotica spp. It is also uncertain that IAP proteins in a pest Diabrotica species can be targeted as a pest control strategy. Furthermore, it is even more uncertain that the expression of such IAP proteins can be modulated using interfering RNA molecules and that if such protein expression can be modulated, whether such modulation will result in toxicity to the target Diabrotica pest.
RNA interference (RNAi) occurs when an organism recognizes double-stranded RNA (dsRNA) molecules and hydrolyzes them. The resulting hydrolysis products are small RNA fragments of about 19-24 nucleotides in length, called small interfering RNAs (siRNAs). The siRNAs then diffuse or are carried throughout the organism, including across cellular membranes, where they hybridize to mRNAs (or other RNAs) and cause hydrolysis of the RNA. Interfering RNAs are recognized by the RNA interference silencing complex (RISC) into which an effector strand (or “guide strand”) of the RNA is loaded. This guide strand acts as a template for the recognition and destruction of the duplex sequences. This process is repeated each time the siRNA hybridizes to its complementary-RNA target, effectively preventing those mRNAs from being translated, and thus “silencing” the expression of specific genes from which the mRNAs were transcribed. Most plant miRNAs show extensive base pairing to, and guide cleavage of their target mRNAs (Jones-Rhoades et al. (2006) Annu. Rev. Plant Biol. 57, 19-53; Llave et al. (2002) Proc. Natl. Acad. Sci. USA 97, 13401-13406). In other instances, interfering RNAs may bind to target RNA molecules having imperfect complementarity, causing translational repression without mRNA degradation. The majority of the animal miRNAs studied so far appear to function in this manner.
There is an ongoing need for compositions and methods for using such compositions having insecticidal activity, for instance for use in crop protection or insect-mediated disease control. Novel compositions are required to overcome the problem of resistance to existing insecticides and/or to help mitigate the development of resistance to existing transgenic plant approaches. Ideally such compositions have a high toxicity and are effective when ingested orally by the target pest and have applicability for use against both the larval and adult stages of the pest insect. Thus any invention which provided compositions in which any of these properties was enhanced would represent a step forward in the art.